Feasibility of whole genome and transcriptome profiling in pediatric and young adult cancers.

The utility of cancer whole genome and transcriptome sequencing (cWGTS) in oncology is increasingly recognized. However, implementation of cWGTS is challenged by the need to deliver results within clinically relevant timeframes, concerns about assay sensitivity, reporting and prioritization of findings. In a prospective research study we develop a workflow that reports comprehensive cWGTS results in 9 days. Comparison of cWGTS to diagnostic panel assays demonstrates the potential of cWGTS to capture all clinically reported mutations with comparable sensitivity in a single workflow. Benchmarking identifies a minimum of 80× as optimal depth for clinical WGS sequencing. Integration of germline, somatic DNA and RNA-seq data enable data-driven variant prioritization and reporting, with oncogenic findings reported in 54% more patients than standard of care. These results establish key technical considerations for the implementation of cWGTS as an integrated test in clinical oncology.

Infections from Body Piercing and Tattoos.

The infectious complications of body piercing and tattooing are reviewed.

The evolving role of neurological imaging in neuro-oncology.

Neuroimaging has played a critical role in the management of patients with neurological disease, since the first ventriculogram was performed in 1918 by Walter Dandy (Mezger et al. Langenbecks Arch Surg 398(4):501-514, 2013). Over the last century, technology has evolved significantly, and within the last decade, the role of imaging in the management of patients with neuro-oncologic disease has shifted from a tool for gross identification of intracranial pathology, to an integral part of real-time neurological surgery. Current neurological imaging provides detailed information about anatomical structure, neurological function, and metabolic and metabolism-important characteristics that help clinicians and surgeons non-invasively manage patients with brain tumors. It is valuable to review the evolution of neurological imaging over the past several decades, focusing on its role in the management of patients with intracranial tumors. Novel neuro-imaging tools and developing technology with the potential to further transform clinical practice will be discussed, as will the key role neurological imaging plays in neurosurgical planning and intraoperative navigation. With increasingly complex imaging modalities creating growing amounts of raw data, validation of techniques, data analysis, and integrating various pieces of imaging data into individual patient management plans, remain significant challenges for clinicians. We thus suggest mechanisms that might ultimately allow for evidence based integration of imaging in the management of patients with neuro-oncologic disease.

Molecular typing of human leukocyte antigen and related polymorphisms following whole genome amplification.

Reliable, high-resolution genotyping of human leukocyte antigen (HLA) polymorphisms is often compromised by DNA samples of suboptimal quality or limited quantity. We tested the feasibility of molecular typing for variants at HLA and neighboring loci using whole genome amplification (WGA) strategy facilitated by the Phi29 DNA polymerase. With little (5-100 ng) starting genomic DNA of varying quality and source materials, WGA was deemed successful in 167 of 169 DNA from 47 cell lines, 100 European Americans, and 22 native Africans. The Phi29-processed DNA provided adequate templates for polymerase chain reaction (PCR)-based analyses of several HLA (A, B, C, DRB1, and DQB1) and related loci (HFE, MICA, and 10 microsatellites) in the 6p24.3-6p21.3 region, with PCR amplicons ranging from 92 to 2200 bp. Five different genotyping techniques resolved and confirmed 364 genotypes when both original and Phi29-processed DNA worked in PCRs. General population genetic analyses provided additional evidence that WGA may represent a reliable and simple approach to securing ample genomic DNA for typing HLA, MICA, and related variants.

Plague.

Plague is a disease that has been present for thousands of years and described since the earliest medical accounts. It occurs today worldwide, and may present in a variety of clinical forms. Bubonic disease, pneumonic plague, and septicemic plague are seen in addition to a number of other less common manifestations. As an agent of bioterrorism,Yersinia pestis could pose an extreme threat if released in the appropriate form and in the appropriate environment. Presumptive diagnosis may be made with readily available techniques, but laboratory handling of specimens requires special care. When there is a strong suspicion of plague, treatment should be instituted immediately, as delaying therapy will result in increased morbidity and mortality.

Surgical treatment of multiple skull abscesses associated with coccidioidomycosis.

This case emphasizes that aggressive neurosurgical management may benefit patients with disseminated coccidioidomycosis and skull abscesses. Disseminated infection due to Coccidioides immitis, the causative agent of coccidioidomycosis, is difficult to treat and often requires prolonged antifungal therapy in addition to surgical debridement. We present a case of a young woman with disseminated coccidioidomycosis who had multiple skull lesions, two of which penetrated the skull and invaded the subgaleal and epidural spaces. Despite prolonged aggressive medical management, these lesions failed to resolve until they were surgically drained.

Human cytomegalovirus infection and expression in human malignant glioma.

Malignant gliomas are the most common primary brain tumors in adults, have no known etiology, and are generally rapidly fatal despite current therapies. Human cytomegalovirus (HCMV) is beta-herpesvirus trophic for glial cells that persistently infects 50-90% of the adult human population. HCMV can be reactivated under conditions of inflammation and immunosuppression, and HCMV gene products can dysregulate multiple cellular pathways involved in oncogenesis. Here we show that a high percentage of malignant gliomas are infected by HCMV and multiple HCMV gene products are expressed in these tumors. These data are the first to show an association between HCMV and malignant gliomas and suggest that HCMV may play an active role in glioma pathogenesis.

Processing liquid-based gynecologic specimens: comparison of the available techniques.

OBJECTIVE: To develop a cytopreparation protocol suitable for satisfactory processing by the AutoCyte PREP System with the gynecologic specimens collected in PreservCyt fluid for the ThinPrep machine. STUDY DESIGN: The residue of a number of gynecologic specimens collected in PreservCyt and processed by ThinPrep were processed by AutoCyte PREP. Some modifications were made in the cytopreparatory protocol in order to obtain satisfactory specimens. RESULTS: The ThinPrep and AutoCyte PREP specimens were examined independently. The results were comparable, with a high degree of concordance between the two techniques. CONCLUSION: Gynecologic specimens collected in PreservCyt and following the ThinPrep specimen collection protocol can be processed using the AutoCyte PREP System. Minor protocol modifications provided comparable diagnostic material. Additional studies are needed to explore the feasibility of this approach and fulfill the various U.S. regulatory agency requirements for the liquid-based Pap test.

Evidence for peroxynitrite-mediated modifications to p53 in human gliomas: possible functional consequences.

Based on previous findings of increased nitric oxide synthase (NOS) expression in human gliomas (4), we hypothesized that peroxynitrite, a highly reactive metabolite of nitric oxide (NO) and superoxide (O(*-)(2)), might be increased in these tumors in vivo. Here we demonstrate that nitrotyrosine (a footprint of peroxynitrite protein modification) is present in human malignant gliomas. Furthermore, we show that p53, a key tumor suppressor protein, has evidence of peroxynitrite-mediated modifications in gliomas in vivo. Experiments in vitro demonstrate that peroxynitrite treatment of recombinant wild-type p53 at physiological concentrations results in formation of higher molecular weight aggregates, tyrosine nitration, and loss of specific DNA binding. Peroxynitrite treatment of human glioma cell lysates similarly resulted in selective tyrosine nitration of p53 and was also associated with loss of p53 DNA binding ability. These data indicate that tyrosine nitration of proteins occurs in human gliomas in vivo, that p53 may be a target of peroxynitrite in these tumors, and that physiological concentrations of peroxynitrite can result in a loss of p53 DNA binding ability in vitro. These findings raise the possibility that peroxynitrite may contribute to loss of wild-type p53 functional activity in gliomas by posttranslational protein modifications.

Repetitive intrathecal injections of poliovirus replicons result in gene expression in neurons of the central nervous system without pathogenesis.

Poliovirus-based vectors (replicons) can be used for gene delivery to motor neurons of the CNS. In the current study, a replicon encoding green fluorescent protein (GFP) was encapsidated into authentic poliovirions, using established procedures. Intrathecal delivery of encapsidated replicons encoding GFP to the CNS of mice transgenic for the human poliovirus receptor did not result in any functional deficits as judged by behavioral testing. Histological analysis of the CNS of mice given a single intrathecal injection of poliovirus replicons encoding GFP revealed no obvious pathogenesis in neurons (or other cell types) within the CNS. The expression of GFP was confined to motor neurons throughout the neuroaxis; a time course of expression of GFP revealed that expression was detectable 24 hr postinoculation and returned to background levels by 120 hr postinoculation. A procedure was devised to allow repetitive inoculation of replicons within the same animal. Behavioral testing of animals that had received 6 to 13 independent inoculations of replicons revealed no functional deficits. Histological analysis of the CNS from animals that had received 6 to 13 sequential inoculations of replicons revealed no obvious abnormalities in neurons or other cell types in the CNS; expression of GFP was demonstrated in neurons 24 to 72 hr after the final inoculation of the replicon. Furthermore, there was no obvious inflammatory response in the CNS after the multiple inoculations. These studies establish the safety and efficacy of replicons for gene delivery to the CNS and are discussed with respect to use of replicons as new therapeutic strategies for spinal cord injuries and/or neurological diseases.

Oxidative stress and heat shock stimulate RGS2 expression in 1321N1 astrocytoma cells.

RGS2, a regulators of G-protein signaling family member, regulates G-protein signaling and is itself controlled in part by regulated expression. We tested if cell stress regulates RGS2 expression in human astrocytoma 1321N1 cells. Treatment with H2O2 increased RGS2 mRNA levels time- and concentration-dependently, with 200 microM H2O2 causing an approximately eightfold increase after 2 h. Peroxynitrite and heat shock also increased RGS2 mRNA levels. H2O2-induced RGS2 expression was negatively regulated by phosphoinositide-3-kinase and extracellular signal-regulated kinases. H2O2 also concentration-dependently increased RGS2 protein levels, and the RGS2 appeared to be predominantly in the nucleus. These results demonstrate that RGS2 expression is up-regulated by cell stress.

Outpatient Management of Infective Endocarditis.

Because of unique host defense characteristics of the endocardium, successful therapy for infective endocarditis (IE) has necessitated bactericidal antimicrobial agents, generally administered in high doses for prolonged periods of time. This has required therapy in the hospital setting. However, in recent years, it has become apparent that outpatient therapy is feasible if appropriate agents can be administered at home, for example, using either home parenteral therapy or oral preparations. Over the past few decades, there has been a trend toward reducing inpatient reimbursement for various conditions, including serious infections. The decision to treat IE on an outpatient basis is made more easily if based on previous published experience. Published reports have concluded that outpatient therapy is more appropriate for "uncomplicated" IE caused by relatively susceptible microorganisms. The purpose of this paper is to review data describing the results of management of IE in the outpatient setting.

Intrasellar cavernous hemangioma. Case report.

The authors present a rare entity, an intrasellar cavernous hemangioma that on neuroimages mimicked a nonfunctioning pituitary macroadenoma in a patient with a known orbital hemangioma. Such lesions can grow extraaxially within the dural sinuses, particularly the cavernous sinus, and present like tumors. A better understanding of the neuroimaging. clinical, and anatomical features of these lesions may prevent difficulties in management.

Second messengers regulate RGS2 expression which is targeted to the nucleus.

Regulators of G-protein Signaling (RGS) proteins attenuate signaling activities of G proteins, and modulation of expression appears to be a primary mechanism for regulating RGS proteins. In human astrocytoma 1321N1 cells RGS2 expression was increased by activation of muscarinic receptors coupled to phosphoinositide signaling with carbachol, or by increased cyclic AMP production, demonstrating that both signaling systems can increase the expression of a RGS family member in a single cell type. Carbachol-stimulated increases in endogenous RGS2 protein levels appeared by immunocytochemical analysis to be largely confined to the nucleus, and this localization was confirmed by Western blot analysis which showed increased nuclear, but not cytosolic, RGS2 after carbachol treatment. Additionally, transiently expressed green fluorescent protein (GFP)-tagged, 6xHis-tagged, or unmodified RGS2 resulted in a predominant nuclear localization, as well as a distinct accumulation of RGS2 along the plasma membrane. The intranuclear localization of GFP-RGS2 was confirmed with confocal microscopy. Thus, RGS2 expression is rapidly and transiently increased by phosphoinositide signaling and by cyclic AMP, and endogenous and transfected RGS2 is largely, although not entirely, localized in the nucleus.

p125 focal adhesion kinase promotes malignant astrocytoma cell proliferation in vivo.

p125 focal adhesion kinase (p125FAK) is a cytoplasmic tyrosine kinase that is activated upon engagement of integrin cell adhesion receptors, and initiates several signaling events that modulate cell function in vitro. To determine the biologic role of p125FAK in malignant astrocytic tumor cells, U-251MG human malignant astrocytoma cells were stably transfected with p125FAK cDNA using the TET-ON system, and stable clones isolated that exhibited an estimated 5- or 20-fold increase in p125FAK expression on administration of 0.1 or 2.0 microg/ml doxycycline, respectively. In vitro studies demonstrated that induction of p125FAK resulted in a 2- to 3-fold increase in cell migration, increased p130CAS phosphorylation, localization of exogenous p125FAK to focal adhesions, and a 2-fold increase in soft agar growth. To determine the role of p125FAK in vivo, clones were injected stereotactically into the brains of scid mice. A 4.5-fold estimated increase in p125FAK expression was induced by administration of doxycycline in the drinking water. Analysis of xenograft brains demonstrated that, upon induction of p125FAK, there was a 1.6- to 2.8-fold increase in tumor cell number, and an increase in mAb PCNA-labeling of tumor cells in the absence of a change in the apoptotic index. Compared to normal brain, the expression of p125FAK was elevated in malignant astrocytic tumor biopsies from patient samples. These data demonstrate for the first time that p125FAK promotes tumor cell proliferation in vivo, and that the underlying mechanism is not associated with a reduction in apoptosis.

The "presyrinx" state: is there a reversible myelopathic condition that may precede syringomyelia?

OBJECT: Alteration of cerebrospinal fluid (CSF) flow has been proposed as an important mechanism leading to the development of syringomyelia. We hypothesize that a "presyrinx" condition due to potentially reversible alteration in normal CSF flow exists and that its appearance may be due to variations in the competence of the central canal of the spinal cord. METHODS: Five patients with clinical evidence of myelopathy, no history of spinal cord trauma, enlargement of the cervical spinal cord with T1 and T2 prolongation but no cavitation, evidence for altered or obstructed CSF flow, and no evidence of intramedullary tumor or a spinal vascular event underwent MR imaging before and after intervention that alleviated obstruction to CSF flow. RESULTS: Preoperatively, all patients demonstrated enlarged spinal cords and parenchymal T1 and T2 prolongation without cavitation. Results of magnetic resonance (MR) imaging examinations following intervention in all patients showed resolution of cord enlargement and normalization or improvement of cord signal abnormalities. In one patient with severe arachnoid adhesions who initially improved following decompression, late evolution into syringomyelia occurred in association with continued CSF obstruction. CONCLUSION: Nontraumatic obstruction of the CSF pathways in the spine may result in spinal cord parenchymal T2 prolongation that is reversible following restoration of patency of CSF pathways. We refer to this MR appearance as the "presyrinx" state and stress the importance of timely intervention to limit progression to syringomyelia.

Effects of a decision support system on physicians' diagnostic performance.

PURPOSE: This study examines how the information provided by a diagnostic decision support system for clinical cases of varying diagnostic difficulty affects physicians' diagnostic performance. METHODS: A national sample of 67 internists, 35 family physicians, and 6 other physicians used the Quick Medical Reference (QMR) diagnostic decision support system to assist them in the diagnosis of written clinical cases. Three sets of eight cases, stratified by diagnostic difficulty and the potential of QMR to produce high-quality information, were used. The effects of using QMR on three measures of physicians' diagnostic performance were analyzed using analyses of variance. RESULTS: Physicians' diagnostic performance was significantly higher (p < 0.01) on the easier cases and the cases for which QMR could provide higher-quality information. CONCLUSIONS: Physicians' diagnostic performance can be strongly influenced by the quality of information the system produces and the type of cases on which the system is used.

Millipore filter cell block preparation: an alternative to cell block in nongynecologic specimens of limited cellularity.

The use of membrane filters to concentrate cytology specimens was first described by Seal (Cancer 1956; 9:866-868). We report on a technique in which a portion of Papanicolaou-stained Millipore filter (Millipore Corp, Bedford, MA) preparation can be converted into a paraffin block for hematoxylin-eosin (H&E) preparations and immunocytochemical analysis (ICC). Seven cases with moderate cellularity and no cell block preparation were retrieved from our cytopathology files. The specimens included: 4 pleural effusions with metastatic adenocarcinoma, and 3 FNA specimens (1 metastatic melanoma, 1 metastatic adenocarcinoma, and 1 thyroid/papillary carcinoma). The filter was removed from the slide, cut in half, and subjected to paraffin embedding in the usual fashion (postfixed in 10% neutral-buffered formalin). Four-micron-thick sections were cut onto Probe-On (Fisher Scientific, Pittsburgh, Pa) slides. ICC was performed using the avidin-biotin-peroxidase complex technique and capillary gap technology. Antibodies included CAM5.2, AE1/AE3, CEA, CD15, LCA, PanCK, S100, HMB45, and thyroglobulin. All cases showed excellent preservation of cellular morphology on H&E. ICC performed on Millipore filter cell block preparations showed specific antibody staining patterns with preservation of cellular details. All antibodies showed their specific staining patterns with clean background and lack of nonspecific staining. This technique has the following advantages: 1) offers an alternative to cell blocks in moderately cellular specimens; 2) clean background; 3) preservation of cytology specimens for future studies.

Endocarditis at the millennium.

The members of the Interplanetary Society (Pus Club) have made significant contributions to the understanding of the pathogenesis of infective endocarditis (IE). Although the incidence of IE has essentially remained unchanged, the spectrum and characteristics of patients potentially affected by this disorder are expanding. Moreover, in addition to the typical microorganisms implicated in IE, there are increasing reports of new or atypical pathogens causing IE, including those that are resistant to standard antibiotic therapy. The infectious diseases community is challenged to continue to provide effective antimicrobial regimens for IE and to further develop diagnostic and surgical strategies to identify and treat patients with this disorder. New information is available regarding the demographics, diagnostic methods, and therapeutic options for the management of IE.